Methods for quantitating individual antibodies from a mixture

ABSTRACT

The present disclosure relates to, inter alia, a method of quantitating an amount of an antibody molecule from a mixture comprising two or more antibody molecules, comprising separating each of the two or more antibody molecules from the mixture by hydrophobic interaction chromatography high performance liquid chromatography (HIC-HPLC) and quantitating an amount of each antibody molecule, wherein the molecular weight of each antibody molecule is within 15 kDa of any other antibody molecule in the mixture and either each antibody molecule is different from another antibody molecule in the mixture by more than about 0.25 unit on the Kyte &amp; Doolittle hydropathy scale or each of the antibody molecules when nm alone on HIC-HPLC elutes at distinct run time with little overlap from the other antibody molecules in the mixture, or both.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/375,887, filed on Aug. 16, 2016, which is incorporated by referenceherein in its entirety.

TECHNICAL FIELD

This disclosure relates to the field of assays for co-formulations oftherapeutic antibodies.

BACKGROUND

Administration of multiple, rather than single, monoclonal antibodies(mAbs) to a patient may improve their diagnostic or therapeuticindication and efficacy. These mAbs may be co-formulated in a singledrug product (DP) and the DP administered to a patient.

A method is required by regulatory agencies to quantitate the individualmAbs in a co-formulated drug substance (cFDS) to be incorporated into aDP, or a DP itself. Developing a method to separate two or more antibodymolecules and to measure the concentration of each mAb is challenging,because the antibody molecules may have similar molecular weights,protein structures, and charge properties.

SUMMARY

This disclosure includes a method of quantitating amounts of antibodiesfrom a mixture comprising a plurality of antibodies. In some aspects,the method may include, among other things, separating each of theplurality of antibodies in the mixture using hydrophobic interactionchromatography high performance liquid chromatography (HIC-HPLC), andquantitating an amount of each antibody in the mixture, wherein amolecular weight of each antibody in the mixture is within 15 kDa of amolecular weight of any other antibodies in the mixture, and either asurface hydrophobicity of each antibody in the mixture is different froma surface hydrophobicity of another antibody in the mixture by more thanabout 0.25 units on the Kyte & Doolittle hydropathy scale, or eachantibody in the mixture, when run on HIC-HPLC individually, elutes at adistinct run time from another antibody in the mixture, or both.

In some embodiments, the surface hydrophobicity of each antibody in themixture is different from the surface hydrophobicity of each otherantibody in the mixture by about 0.5 to about 1.0 units on the Kyte &Doolittle hydropathy scale. In further embodiments, the surfacehydrophobicity of each antibody in the mixture is determined bycalculating surface hydrophobicity based on protein structure orstructural model, rapid screening for solubility in ammonium sulfate orPEG8000, or rapid screening for molecule interaction by affinitycapture-self-interaction nanoparticle spectroscopy (AC-SINS).

In additional embodiments, a first antibody in the mixture elutes at afirst run time during a HIC-HPLC run, a second antibody in the mixtureelutes at a second run time during the HIC-HPLC run, and the first andsecond run times do not overlap. In yet further embodiments, a firstantibody in the mixture and a second antibody in the mixture haveprotein sequences that are at least 90% homologous, the first antibodyand the second antibody have protein structures that are at least 90%homologous, as determined by their protein sequences, or the firstantibody and the second antibody have isoelectric points (pI) withinabout 0.6 of one another, as determined by their protein sequences.

In some embodiments, the plurality of antibodies comprises threeantibodies. In further embodiments, one or more of the antibodies in themixture are monoclonal antibodies. In still further embodiments, one ormore of the antibodies in the mixture are human monoclonal antibodies.In other embodiments, two or more of the antibodies in the mixture areof the same isotype. In some embodiments, two or more of the antibodiesin the mixture are variants of each other. In further embodiments, twoor more of the antibodies in the mixture bind to the same antigen.

In some embodiments, the mixture is a co-formulated composition. Inadditional embodiments, the co-formulated composition is configured totreat MERS in a human patient. In further embodiments, the co-formulatedcomposition is configured to treat Ebola hemorrhagic fever in a humanpatient. In further embodiments, the co-formulated composition isconfigured to treat macular degeneration in a human patient. In yetfurther embodiments, the two or more antibodies in the co-formulatedcomposition are configured to treat an infectious disease in a humanpatient. In some embodiments, the co-formulated composition is includedin a drug product.

In some embodiments, the HIC-HPLC is performed in a buffer at about pH5.0 to about pH 7.0. In further embodiments, the method furthercomprises generating a chromatograph from the HIC-HPLC, wherein forelution of each antibody in the mixture, the chromatograph shows a peakthat does not overlap with other peaks in the chromatograph.

This disclosure also includes a method of quantitating amounts ofantibodies from a mixture comprising a plurality of antibodies, themethod comprising: separating each of the plurality of antibodies in themixture using hydrophobic interaction chromatography high performanceliquid chromatography (HIC-HPLC), wherein a molecular weight of eachantibody in the mixture is within 15 kDa of a molecular weight of eachother antibody in the mixture; quantitating an amount of each antibodyin the mixture; and generating a chromatograph from the HIC-HPLC,wherein for elution of each antibody in the mixture, the chromatographshows a peak that does not overlap with other peaks in thechromatograph. In some embodiments, one or more of the plurality ofantibodies are human monoclonal antibodies. In further embodiments,either a surface hydrophobicity of each antibody in the mixture isdifferent from a surface hydrophobicity of another antibody in themixture by more than about 0.25 units on the Kyte & Doolittle hydropathyscale, or each antibody in the mixture, when run on HIC-HPLCindividually, elutes at a distinct run time from another antibody in themixture, or both.

Numerous other aspects and embodiments are provided in accordance withthese and other aspects of the disclosure. Other features and aspects ofthe present disclosure will become more fully apparent from thefollowing detailed description and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate various examples and togetherwith the description, serve to explain the principles of the presentdisclosure. Any features of an embodiment or example described herein(e.g., device, method, etc.) may be combined with any other embodimentor example, and are encompassed by the present disclosure.

FIG. 1 shows an exemplary chromatograph of a HIC-HPLC run of aco-formulation comprising anti-Ebola mAbs.

FIGS. 2-8 show exemplary chromatographs of HIC-HPLC runs of aco-formulation comprising anti-MERS mAbs.

FIG. 9 shows an exemplary chromatograph of a HIC-HPLC run of aco-formulation comprising anti-Ebola mAbs.

FIG. 10 is a plot of linearity of HIC-HPLC, plotting peak area againstthe amount of individual anti-Ebola mAb run.

FIG. 11 is a plot of range, plotting percent recovery of the mAbsagainst the amount of individual anti-Ebola mAb run.

FIG. 12 is a plot of the accuracy of HIC-HPLC with different mixturesample lots, plotting percent recovery of the mAbs against the amount ofindividual anti-Ebola mAb run.

FIG. 13 is a plot of the accuracy of HIC-HPLC with different ratios ofindividual anti-Ebola mAbs, plotting percent recovery of the mAbsagainst the amount of individual anti-Ebola mAb run.

FIG. 14 is a plot of storage stability of the mAbs in the cFDS, plottingamount of individual anti-Ebola mAbs against storage time in months.

FIG. 15 shows an exemplary chromatograph of a cation exchange ultra highpressure liquid chromatography (CEX-UPLC) run of a co-formulationcomprising anti-Ebola mAbs.

FIG. 16 is a plot of linearity of CEX-UPLC, plotting peak area againstamount of individual anti-Ebola mAb.

FIG. 17 shows an exemplary chromatograph of a size-exclusion ultra highpressure liquid chromatography (SE-UPLC) run of a co-formulationcomprising anti-Ebola mAbs.

FIG. 18 shows an exemplary imaged capillary isoelectric focusing (iCIEF)profile of a co-formulation comprising anti-Ebola mAbs.

FIG. 19 shows an exemplary chromatograph of a reverse phase ultra highpressure liquid chromatography (RP-UPLC) run of a co-formulationcomprising anti-Ebola mAbs.

DETAILED DESCRIPTION

The term “antibody” is sometimes used interchangeably with the term“immunoglobulin.” Briefly, it may refer to a whole antibody comprisingtwo light chain polypeptides and two heavy chain polypeptides. Wholeantibodies include different antibody isotypes including IgM, IgG, IgA,IgD, and IgE antibodies. The term “antibody” may include, for example, apolyclonal antibody, a monoclonal antibody (mAb), a chimerized orchimeric antibody, a humanized antibody, a primatized antibody, adeimmunized antibody, and a fully human antibody. The antibody may bemade in or derived from any of a variety of species, e.g., mammals suchas humans, non-human primates (e.g., orangutan, baboons, orchimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits,guinea pigs, gerbils, hamsters, rats, and mice. The antibody may be apurified or a recombinant antibody. The antibody can also be anengineered protein or antibody-like protein containing at least oneimmunoglobulin domain (e.g., a fusion protein). The engineered proteinor antibody-like protein may also be a bi-specific antibody or atri-specific antibody, or a dimer, trimer, or multimer antibody, or adiabody, a DVD-Ig, a CODV-Ig, an Affibody®, or a Nanobody®.

The terms “variant of an antibody,” “antibody variant,” and the like,refer to an antibody that varies from another antibody in that thevariant antibody is a deletion variant, insertion variant, and/orsubstitution variant of the other antibody.

The term “human antibody,” as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. Human mAbs may include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations introduced by random or site-specific mutagenesis in vitro orby somatic mutation in vivo), for example in the CDRs and in particularCDR3. However, the term “human antibody,” as used herein, is notintended to include mAbs in which CDR sequences derived from thegermline of another mammalian species (e.g., mouse), have been graftedonto human FR sequences. The term includes antibodies recombinantlyproduced in a non-human mammal, or in cells of a non-human mammal. Theterm is not intended to include antibodies isolated from or generated ina human subject.

As used herein, the terms “treat,” “treating,” or “treatment” refer tothe reduction or amelioration of the severity of at least one symptom orindication of a disease or condition due to the administration of aco-formulation of two or more antibodies to a subject in need thereof.The terms include inhibition of progression of disease. The terms alsoinclude positive prognosis of disease.

The terms “prevent,” “preventing” or “prevention” refer to inhibition ofmanifestation of a disease or condition any symptoms or indications ofthat disease or condition upon administration of a co-formulation of twoor more antibodies.

For the terms “for example” and “such as,” and grammatical equivalencesthereof, the phrase “and without limitation” is understood to followunless explicitly stated otherwise. As used herein, the term “about” andthe signifier “˜” are meant to account for variations due toexperimental error. All measurements reported herein are understood tobe modified by the term “about,” whether or not the term is explicitlyused, unless explicitly stated otherwise. As used herein, the singularforms “a,” “an,” and “the” include plural referents unless the contextclearly dictates otherwise.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Antibody molecules, such as monoclonal antibody molecules, may beco-formulated to treat one or more diseases or conditions in a patient(including a human patient). The terms “patient” and “subject” are usedinterchangeably herein.

A co-formulated drug product (DP) may include a co-formulated drugsubstance (cFDS) (also referred to herein as a co-formulation)containing two or more (e.g., three) human monoclonal antibody (mAb)molecules. The cFDS is prepared by mixing purified mAbs at apredetermined ratio. A method is required by regulatory agencies toquantitate each of individual mAbs in the cFDS.

The mAb molecules in the co-formulation may be similar to each other:they may be immunoglobulins (such as IgG1) with about the same molecularweight (e.g., ˜145 kDa); with similar protein structure and chargeproperties.

Methods disclosed herein for quantitating similar mAb molecules in aco-formulation are precise, accurate, reproducible, suitable for use inquality control environments, do not use expensive equipment, and do notrequire cumbersome sample preparation.

Methods

This disclosure provides methods of quantitating an amount of anantibody molecule from a mixture comprising two or more antibodymolecules. The method may comprise separating each of the two or moreantibody molecules from the mixture by hydrophobic interactionchromatography high performance liquid chromatography (HIC-HPLC) andquantitating an amount of each antibody molecule, wherein the molecularweight of each antibody molecule is within 15 kDa of any other antibodymolecule in the mixture and either each antibody molecule is differentfrom another antibody molecule in the mixture by more than about 0.25units on the Kyte & Doolittle hydropathy scale (see, e.g., Kyte andDoolittle, J. Mol. Biol. 157, 105-132 (1982)), or each of the antibodymolecules, when run alone on HIC-HPLC, elutes at a distinct run timewith little overlap from other antibody molecules in the mixture, orboth. In certain embodiments, for example, the relative hydrophobicityof each antibody molecule is different from each other antibody moleculein the mixture by about 0.5 to about 1.0 unit on the Kyte & Doolittlehydropathy scale. In certain other embodiments, each of the antibodymolecules, when run alone on HIC-HPLC, elutes at a distinct run timewith little overlap from the other antibody molecules in the mixture. Incertain embodiments, the mixture comprises a plurality of antibodymolecules (e.g., two, three, four, or five antibody molecules).

If two antibody molecules from the mixture, when run on HIC-HPLC, havesignificant overlap in the chromatograph monitoring elution profiles ofthe antibody molecules by absorbance verses time of elution, thisindicates that the two antibody molecules elute at times that are close,resulting in a lack of separation within the column such that theantibody molecules are not purified (e.g., less than 50% pure). In suchcases, the individual antibodies cannot be fully quantitated because ofthat significant overlap. In some embodiments, two antibody moleculesfrom the mixture, when run on HIC-HPLC, have little to no overlap in thechromatograph monitoring elution profiles of the antibody molecules byabsorbance verses time of elution, in that the antibody molecules eluteat times that are not close, resulting in separation such that theantibody molecules are substantially purified, each being at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure.

In some embodiments, methods disclosed herein are used to quantitate anindividual population of antibodies, also referred to as “an antibodymolecule,” from a mixture of antibodies, which includes two or moredifferent populations of antibodies, where the different populationshave at least one amino-acid difference between them. In someembodiments, a population of antibodies may have the same amino acidsequence as that of another population in the mixture of antibodies, butthey may differ in post-translational modifications. In certainembodiments, a mixture is a co-formulated composition, such as orincluding a co-formulated drug substance (cFDS). In certain embodiments,a mixture comprises excipients. In certain embodiments, a mixturecomprises sucrose, and may be a sucrose drug substance comprising mAbmolecules. Each antibody in the mixture of antibodies will have asimilar molecular weight, within 1, 2, 3, 4, 5, 10, or 15 kilodaltons(kDa) of each other. In another aspect, the average molecular weight ofall the antibodies will be about 150 kDa. This method can also be usedwith two or more trap molecules that have similar molecular weights. Incertain further embodiments, the co-formulated composition comprisesVEGF-Trap. (See, e.g., U.S. Pat. No. 9,265,827 and U.S. patentapplication Ser. No. 14/943,490, which are incorporated by referencehere in their entireties.) This method may also be used to monitor theconcentration for each of the mAbs in a mixture (e.g., a co-formulation)in monitoring storage stability. In certain embodiments, this method isused to separate antibody molecules that cannot be separated by anotherchromatographic method, such as by reverse phase high pressure liquidchromatography (HPLC) or ultra high pressure liquid chromatography(UPLC).

Hydrophobic Interaction Chromatography (HIC) separates antibodies in adecreasing salt gradient, based on differences in surface hydrophobicityof the antibodies. Separation using HIC is based on the reversibleinteraction between an antibody and the hydrophobic ligand bound to thechromatography matrix. Though hydrophobic amino acids of proteins andpeptides are usually located away from molecular surfaces, biomoleculeshave some hydrophobic groups that are exposed to allow interaction withhydrophobic ligands on media. The hydrophobic interaction is enhanced bybuffers with high ionic strength.

Any suitable HIC-HPLC column may be employed for methods disclosedherein, including, without limitation, Dionex ProPac HIC-10, mAbPacHIC-10, mAbPac HIC-20, mAbPac HIC-Butyl (Dionex, Thermo FisherScientific, Sunnyville, Calif.). The buffer may be any suitable buffer.In certain embodiments, the HIC-HPLC is performed in a buffer pH betweenabout pH 5.0 to about pH 7.0, or between about pH 6.0 to about pH 7.0,or between about 6.5 to about 7.5. In certain embodiments, the columnsare Dionex MabPac HIC-10 (100×4.6 mm, 5 μm, Pore Size 1000A°) and ProPacHIC-10 (100×4.6 mm, 5 μm, Pore Size 300A°).

A mixture comprising two or more antibodies is run on HIC-HPLC. Theantibodies are eluted at separate times (run times). The antibodies aremonitored by measuring their absorbance at, e.g., 280 nm on the UVspectrum. A chromatograph may be used to monitor and to document therun. In certain embodiments, buffers used in the HIC-HPLC are differentgradients of 1M ammonium sulfate (from 100% to 0%) in 100 mM phosphatebuffer.

In certain embodiments, the antibody molecules are quantitated bycomparing their absorbance at 280 nm to a standard curve. A standardcurve may be constructed by determining the absorbance of a knownantibody at known concentrations from a HIC-HPLC run, at an absorbanceof, for example, 280 nm. The greater the absorbance (or “opticaldensity”), the higher the protein concentration. These data for knownconcentrations of an antibody are used to make the standard curve,plotting concentration on the X axis, and the absorbance obtained fromthe chromatograph of the elution profile of the antibody from theHIC-HPLC run on the Y axis. A sample comprising two or more antibodiesof unknown concentrations, including the antibody from the standardcurve, are run. To analyze the data, the antibodies are separated byHIC-HPLC. The elution profiles of the antibodies are displayed on achromatograph, with antibody concentration represented by absorbancemonitored over time by a spectrophotometer. The absorbance of eachantibody, as identified by its elution time (run time) is used to locatea corresponding absorbance value on the standard curve. Thecorresponding X-axis value for that point on the standard curve is theconcentration of that antibody in the sample.

In some aspects of the current disclosure, methods for quantitatingindividual antibodies in a mixture disclosed herein may be useful if therelative surface hydrophobicity of each antibody in the mixture isdifferent from that of each other antibody in the mixture by greaterthan about 0.25 units, such as by about 0.5 to about 1.0 units, on theKyte & Doolittle hydropathy scale. The relative surface hydrophobicityof the antibodies may be determined/estimated by a number of methods.

For example, HIC-HPLC may be used to determine the relative surfacehydrophobicity of different antibody molecules. Each of the antibodymolecules is run alone on HIC-HPLC and the run time (elution time) foreach antibody is determined.

In certain embodiments, the difference in surface hydrophobicity betweenantibodies in a mixture is determined by one or more of the followingmethods: calculated surface hydrophobicity based on protein structure orstructural model, rapid screening method for solubility in ammoniumsulfate or PEG8000, and rapid screening for molecule interaction byaffinity capture—self-interaction nanoparticle spectroscopy (AC-SINS).

The relative surface hydrophobicity may be calculated based on knownstructure of the antibody (based on, for example, crystal structure orNMR structure) or a structural model. Such computational methods areknown in the art.

The relative surface hydrophobicity may be calculated/estimated bydetermining the solubility of the antibody molecules by, for example,rapid screening method for solubility in conditions that enhancehydrophobicity, such as in ammonium sulfate or PEG8000. The more solublea protein, the less hydrophobic. (See, e.g., Kramer et al., BiophysicalJournal Volume 12 Apr. 1907-1915 (2012).)

The relative surface hydrophobicity may be calculated/estimated by rapidscreening for molecule interaction by affinity capture—self-interactionnanoparticle spectroscopy (AC-SINS). (See, e.g., Estep et al., mAbs,7:3, 553-561 (2015).) AC-SINS is an approach that coats goldnanoparticles with polyclonal anti-human antibodies, uses theseconjugates to immobilize human mAbs, and evaluates mAb self-interactionsby measuring the plasmon wavelengths of the antibody conjugates as afunction of ammonium sulfate concentration.

In certain other embodiments, antibodies in a mixture that can beseparated using the described methods will have similar size or overallcharge as determined by one or more of the following methods: proteinstructure, or structural modeling, based on sequence and other knownprotein structures, calculated overall protein charge property based onprotein sequences, calculated hydrophobicity based on protein sequences,and protein sequencing.

In certain embodiments, antibodies in a mixture have at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, or at least 99%homologous protein structure or structural models based on sequence andother known protein structures. In certain embodiments, antibodies in amixture have calculated protein charge properties within 3, 4, or 5, 10or 15 elementary units of one another, where protein charge propertiesare calculated based on protein sequence. In certain embodiments, two ormore antibodies in a mixture have at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or at least 99% identity between theirprotein sequences.

In an aspect, methods of quantifying antibodies in a mixture usingHIC-HPLC as disclosed herein may not be as useful if there is a largedifference between the antibody molecules as calculated or estimated byprotein structure, sequence-based structural models, protein sequence,and other known aspects of protein structure. If there are differencesin the hydrophobic profiles of the antibodies, then methods usingHIC-HPLC may nevertheless facilitate separation of the antibody species.

In another aspect, methods of quantifying antibodies in a mixture usingHIC-HPLC as disclosed herein may not be as useful if there is a largedifference between the antibody molecules calculated/estimated by thecalculated overall protein charge property of each antibody molecule,based on the protein sequence of each antibody molecule. If there aredifferences in the hydrophobic profiles of the antibodies, then methodsusing HIC-HPLC may nevertheless facilitate separation of the antibodyspecies.

In another aspect, methods of quantifying antibodies in a mixture usingHIC-HPLC as disclosed herein may not be as useful if there are largedifferences between the antibody molecules calculated/estimated byinspecting the protein charge or size. If there are differences in thehydrophobic profiles of the antibodies, then methods using HIC-HPLC maynevertheless facilitate separation of the antibody species.

In certain embodiments, one or more of the antibodies in a mixture ofantibodies are monoclonal antibodies. In certain embodiments, one ormore of the monoclonal antibodies are human monoclonal antibodies. Incertain embodiments, two or more of the antibody molecules are of thesame isotype. In certain embodiments, two or more of the antibodymolecules are variants of each other. In yet certain other embodiments,two or more of the antibody molecules bind to the same antigen. The mAbsmay be whole antibody molecules.

In certain embodiments, the mixture is a co-formulated composition. Incertain further embodiments, the co-formulated composition comprises twoor more mAbs that are effective for treating Middle East RespiratorySyndrome (MERS) in a human patient. Antibodies to the MERS corona virus(MERS-CoV) are disclosed in, for example, U.S. patent application Ser.No. 14/717,760 and International Application Publication No.WO2015/179535A1. In certain further embodiments, the co-formulatedcomposition comprises mAbs that are effective in treating Ebolahemorrhagic fever in a human patient. Antibodies to the Ebola virus aredisclosed in, for example, U.S. patent application Ser. No. 15/005,334,filed on Jan. 25, 2016. In certain further embodiments, theco-formulated composition comprises mAbs that are effective in treatingmacular degeneration in a human patient. In certain further embodiments,the co-formulated composition comprises mAbs alirocumab and evinacumabdisclosed in U.S. Provisional Application No. 62/302,907. In certainembodiments, the co-formulated composition comprises PD1 antibodies andother immune-oncology antibody products, such as bispecific antibodies.(See, e.g., disclosure of PD-1 and CD3×CD20 in U.S. ProvisionalApplication No. 62/270,749, U.S. patent application Ser. No. 15/386,443,and U.S. patent application Ser. No. 15/386,453; and PD-1 and Lag3 inU.S. Provisional Application 62/365,006 and U.S. patent application Ser.No. 15/289,032.) In certain embodiments, the co-formulated compositioncomprises anti-Zika virus antibodies. (See, e.g., U.S. ProvisionalApplication No. 62/363,546.) In certain further embodiments, theco-formulated composition comprises trevogrumab and Activin Aantibodies. (See, e.g., U.S. Pat. No. 8,871,209.) In certainembodiments, two or more antibodies in the co-formulated composition maytreat an infectious disease in a human patient. The disclosure of eachof the above-cited patent documents in this paragraph is herebyincorporated by reference.

The anti-MERS mAbs in a co-formulation may bind to, for example, thespike protein of MERS-CoV (e.g., the spike protein of MERS-CoV isolateEMC/2012). The spike protein's epitope may be within the receptorbinding domain of the spike protein (e.g., amino acids selected from theamino acids 367 to 606 of GenBank Accession No. AFS88936.1). Theanti-MERS mAbs in the co-formulated composition may be: a fully humanmonoclonal antibody that binds to the MERS-CoV spike protein; one thatinteracts with one or more amino acid residues in the receptor bindingdomain of the MERS-CoV spike protein selected from amino acid residues367 to 606 of GenBank Accession No. AFS88936.1; one that binds toMERS-CoV spike protein with a dissociation constant (Ko) of less than18.5 nM, as measured in a surface plasmon resonance assay; or one thatblocks binding of MERS-CoV spike protein to dipeptidyl peptidase 4(DPP4) by more than 90%.

The co-formulated composition may be any composition comprising two ormore antibodies directed to the same or different target, and areeffective in treating the same or different disease or condition in apatient, including a human patient.

Pharmaceutical Compositions and Formulations

Compositions containing two or more antibody molecules may be formulatedas a pharmaceutical composition (e.g., a DP) for administering to asubject. The pharmaceutical compositions can include, for example, threeantibody molecules. Any suitable pharmaceutical compositions andformulations, as well as suitable methods for formulating and suitableroutes and suitable sites of administration, are within the scope ofthis invention. Also, unless otherwise stated, any suitable dosage(s)and frequency of administration are contemplated.

The mAbs in the co-formulations are purified by methods known in the artbefore being co-administered. The co-formulations of two or moreantibodies may be any suitable co-formulations.

The pharmaceutical compositions/co-formulations may include apharmaceutically acceptable carrier (i.e., an excipient). A“pharmaceutically acceptable carrier” refers to, and includes, any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, diluents, glidants,etc. The compositions can include a pharmaceutically acceptable salt,e.g., an acid addition salt or a base addition salt (see e.g., Berge etal. J Pharm Sci 66:1-19 (1977)). The composition can include sucrose orcan be coated when appropriate.

In certain embodiments, the protein compositions can be stabilized andformulated as a solution, microemulsion, dispersion, liposome,lyophilized cake, solid, etc. Sterile injectable solutions can beprepared by incorporating two or more mAbs in the required amounts in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating two or more mAb molecules intoa sterile vehicle that contains a basic dispersion medium and therequired other ingredients from those enumerated above. The properfluidity of a solution can be maintained, for example, by the use of acoating such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of surfactants. Prolongedabsorption of injectable compositions can be brought about by includingin the composition a reagent that delays absorption, for example,monostearate salts, and gelatin.

EXAMPLES

For this invention to be better understood, the following examples areset forth. These examples are for purposes of illustration only and arenot be construed as limiting the scope of the invention in any manner.

The anti-Ebola mAbs in the co-formulation used in Examples 1 and 3-7 arewhole, fully human IgG 1 monoclonal antibodies. The three mAbs (mAb A,mAb B, and mAb C) have similar molecular weights (e.g., about 145 kDa),protein structure, and charge properties (e.g., a difference in pI ofabout 0.6 or less, as determined by protein sequences). The isoelectricpoint (pI) of mAb A is determined to be 9.0, the pI of mAb B isdetermined to be 8.5, and the pI of mAb C is determined to be 9.1.

The anti-MERS mAbs in the co-formulation used in Example 2 are whole,fully human anti-Ebola mAbs. The two mAbs have similar molecular weights(e.g., within about 15 kDa of one another) and charge properties. (SeeU.S. Patent Publication No. US2015/0337029, WO2015/179535A1, and U.S.patent application Ser. No. 14/717,760, the disclosure of each of whichis hereby incorporated by reference herein.)

Example 1

A HIC-HPLC method is used to quantitate 3 anti-Ebola monoclonalantibodies (mAb 1, mAb 2, and mAb 3) of similar molecular weights,protein structures, and charge properties from a co-formulation by firstseparating the 3 mAbs from the co-formulation and then quantitating eachof them.

A Dionex ProPac HIC-10 column is used, Cat#063655 (Dionex, Thermo FisherScientific, Sunnyville, Calif.), 4.6×100 mm.

Preparation of Mobile Phases

The mobile phases include a Mobile Phase A and a Mobile Phase B. MobilePhase A includes 1M Ammonia phosphate and 100 mM phosphate, at a pH of7.0. Preparation of Mobile Phase A includes: dissolving 13.8 g of sodiumphosphate monobasic, monohydrate (NaH₂PO₄.H₂O) and 132.1 g of ammoniaphosphate in 800 mL Milli Q; adjusting the pH to 7.0 with 50% NaOH;bringing the volume to 1000 mL; and filtering the solution through a0.22 μM filter. Mobile Phase B includes 100 mM phosphate at a pH of 7.0.Preparation of Mobile Phase B includes: Dissolving 13.8 g of sodiumphosphate monobasic, monohydrate (NaH₂PO₄.H₂O) in 900 mL Milli Q;adjusting the pH to 7.0 with 50% NaOH; bringing the volume to 1000 mL;and filtering the solution through a 0.22 μM filter.

HIC-HPLC Method

The HIC-HPLC is run through the aforementioned column at a flow rate of0.5 mL/minute. The column temperature is kept at 30° C., and theco-formulation sample temperature is 5° C. The stop time for the columnis 40 minutes. The effluent's absorbance of 280 nm ultraviolet light ismonitored using a UV detector. Table 1 below shows the mix of MobilePhase A and Mobile Phase B as percentages of the mobile phasecomposition gradient to be introduced over the column run time.

TABLE 1 Mobile phase gradient Time (min) % Mobile Phase A % Mobile PhaseB 0 60 40 4 60 40 19 0 100 32 0 100 33 60 40 40 60 40

Calibration with Three Individual mAbs

In order to calibrate results for the three individual mAbs, knownamounts of each mAb in FDS are run. A FDS with a known concentration ofeach mAb is prepared, and the concentration is measured by eitherreverse phase (RP) chromatography or Solo VPE® Spectroscopy (e.g., CTechnologies, Inc.). The known FDS for each mAb are as follows:

TABLE 2 Concentration of each mAb FDS as measured by RP or Solo VPE ®Known mAb Known mAb concentration Formulation amount (g) (t = 0) (mg/mL)mAb 1 FDS 84.08 51.31 (by Solo VPE ®) mAb 2 FDS 84.13 53.40 (by RP) mAb3 FDS 84.19 50.65 (by RP)

A control sample is also prepared for the calibration sequence. Thecontrol sequence has a concentration of 50.31 mg/mL as measured by SoloVPE® Spectroscopy (e.g., C Technologies, Inc.). One injection of thecontrol sample is 12.0 μL (603.72 μg).

Table 2 below lists the calibration samples used of each of the mAb FDS.

TABLE 2 Calibration samples Amount Number of injections mAb 1 FDS: 50.5mg/mL working Standard Lot# YC10190-42A (Regeneron Pharmaceuticals,Tarrytown, NY) 2.0 μL = 101 μg 1 injection 3.0 μL = 151.5 μg 1 injection4.0 μL = 201 μg 1 injection 5.0 μL = 252.5 μg 1 injection 6.0 μL = 303μg 1 injection mAb 2 FDS: 51.7 mg/mL working Standard Lot# YC10190-42B(Regeneron Pharmaceuticals, Tarrytown, NY) 2.0 μL = 103.4 μg 1 injection3.0 μL = 155.1 μg 1 injection 4.0 μL = 206.8 μg 1 injection 5.0 μL =258.5 μg 1 injection 6.0 μL = 310.2 μg 1 injection mAb 3 FDS: 51.4 mg/mLworking Standard Lot# YC10190-42C (Regeneron Pharmaceuticals, Tarrytown,NY) 2.0 μL = 102.8 μg 1 injection 3.0 μL = 154.2 μg 1 injection 4.0 μL =205.6 μg 1 injection 5.0 μL = 257 μg 1 injection 6.0 μL = 308.4 μg 1injection

The calibration sequence is run. During the calibration sequence, oneinjection of the control sample is introduced at the beginning of eachsample set (e.g., the set of mAb 1 FDS injections, the set of mAb 2 FDSinjections, or the set of mAb 3 FDS injections), after every 20-24injections of mAb FDS, and at the end of each injection sequence.

During one calibration sequence, one injection of each known amount ofmAb is performed. During another calibration sequence, four injectionsof each known amount of mAb are performed to determine repeatability ofinjections.

A standard calibration curve is constructed for each mAb. Thecalibration curve for each mAb has an R²≥0.999, and the variability ofrepeated injections of the same amounts of each mAb is ≤1%.

Control Specifications

A control HIC-HPLC run of each known mAb FDS is performed. Percentrecovery for each known mAb FDS is calculated by measuring theconcentration by HIC-HPLC, and then dividing the concentration measuredby HIC-HPLC by the known mAb concentration in Table 2. Percent recoveryis calculated to be 90-110% of the known concentrations for all threeknown mAb FDS.

Unknown Samples

A sample co-formulation having a total of 600 μg mAb (e.g., 12 μL of a50 mg/mL mAb sample) is run through HIC-HPLC. For example, 12 μL of aco-formulation including 600 μg total of mAb may include the equivalentof three injections of 200 μg/injection of individual mAb. Theindividual mAbs in the co-formulation are mAb 1, mAb 2, and mAb 3.

A duplicate run of the sample is performed, and the averageconcentration (mg/mL) of each individual mAb in the co-formulation andthe ratio of each mAb to the total amount of mAb is reported.

FIG. 1 is a chromatograph showing an HIC-HPLC run of thisco-formulation. Each mAb is depicted by a separate line, and the cFDS asa whole is also depicted by a line. The 3 antibodies are well separatedby HIC-HPLC, with little overlap in peaks.

Example 2

Multiple HIC-HPLC methods are used to quantitate 2 anti-MERS monoclonalantibodies of similar molecular weights and charge properties from aco-formulation by first separating the 2 mAbs from the co-formulationand then quantitating each of them.

mAb Drug Substances

Two mAb (mAb 1 and mAb 2) drug substances are combined. mAb 1 drugsubstance includes 52.3 mg/ml mAb 1 and 10 mM His, at a pH of 5.5(Regeneron Pharmaceuticals, Tarrytown, N.Y.). mAb 2 drug substanceincludes 40.4 mg/ml mAb 2 and 10 mM His, at a pH of 6.0, and includes 5%(w/v) sucrose (Regeneron Pharmaceuticals, Tarrytown, N.Y.).

HIC-HPLC Methods

Seven HIC-HPLC methods are run. In each method, 50 μg (mAb 1 and mAb 2)of sample is loaded. The effluent is monitored using UV detection at 280nm. Two solvents are used, in varying proportion, in the mobile phase.Solvent A is 1M (NH₄)SO₄ 20 mM Acetate at pH 5.5, and Solvent B is 20 mMAcetatae at pH 5.5.

1. Method 1

TABLE 3.1 Method 1 gradient and flow information Flow Time (min)(mL/min) % A in mobile phase % B in mobile phase Startup 0.500 100 0 40.500 100 0 29 0.500 0 100 40 0.500 0 100 41 0.500 100 0 50 0.500 100 0

FIG. 2 shows a chromatograph of this run. The two antibodies aresomewhat separated but have significant overlap.

2. Method 2—0.5% per minute

TABLE 3.2 Method 2 gradient and flow information Flow Time (min)(mL/min) % A in mobile phase % B in mobile phase Startup 0.500 40 60 40.500 40 60 40 0.500 20 80 41 0.500 0 100 51 0.500 0 100 52 0.500 40 6056 0.500 40 60

FIG. 3 shows a chromatograph of this run. The two antibodies aresomewhat separated but have significant overlap.

3. Method 3—0.2% per minute

TABLE 3.3 Method 3 gradient and flow information Flow Time (min)(mL/min) % A in mobile phase % B in mobile phase Startup 0.500 39 61 40.500 39 61 49 0.500 39 70 50 0.500 0 100 60 0.500 0 100 61 0.500 39 6165 0.500 39 61

FIG. 4 shows a chromatograph of this run. The two antibodies aresomewhat separated but have significant overlap.

4. Method 4—2% per minute

TABLE 3.4 Method 4 gradient and flow information Flow Time (min)(mL/min) % A in mobile phase % B in mobile phase Startup 0.500 100 0 40.500 100 0 34 0.500 40 60 35 0.500 0 100 45 0.500 0 100 46 0.500 100 050 0.500 100 0

FIG. 5 shows a chromatograph of this run. The two antibodies aresomewhat separated but still have some overlap.

5. Method 5—1% per minute

TABLE 3.5 Method 5 gradient and flow information Flow Time (min)(mL/min) % A in mobile phase % B in mobile phase Startup 0.500 80 20 40.500 80 20 34 0.500 50 50 35 0.500 0 100 45 0.500 0 100 46 0.500 80 2050 0.500 80 20

FIG. 6 shows a chromatograph of this run. The two antibodies aresomewhat separated but have some overlap.

6. Method 6—1% per min

TABLE 3.6 Method 6 gradient and flow information Flow Time (min)(mL/min) % A in mobile phase % B in mobile phase Startup 0.500 90 10 40.500 90 10 44 0.500 50 50 45 0.500 0 100 55 0.500 0 100 56 0.500 90 1060 0.500 90 10

FIG. 7 shows a chromatograph of this run. The two antibodies aresomewhat separated but have some overlap.

7. Method 7—0.5% per minute

TABLE 3.7 Method 7 gradient and flow information Flow Time (min)(mL/min) % A in mobile phase % B in mobile phase Startup 0.500 87 130 40.500 87 13 74 0.500 52 48 75 0.500 0 100 85 0.500 0 100 86 0.500 87 1390 0.500 87 13

FIG. 8 shows a chromatograph of this run. The two antibodies aresomewhat separated but have some overlap.

Example 3

HIC separation of a co-formulation comprising 3 anti-Ebola mAbs ofsimilar molecular weights, protein structures, and charge properties isdeveloped and evaluated on Agilent 1100 HPLC system.

The HIC-HPLC method uses a Dionex ProPac HIC-10 column with a two-partmobile phase (including Mobile Phase A (100 mM phosphate, 1 M ammoniumsulfate, pH 7.0) and Mobile Phase B (100 mM phosphate, pH 7.0)) at aflow rate of 0.5 mL/minute. The HIC method is qualified using afive-point calibration curve for each mAb. The accuracy (% recovery) isdetermined by comparing the measured concentration to the theoreticalconcentration of each mAb. A gradient of 600 to 0 mM ammonium sulfate isused to run the column.

FIG. 9 shows a chromatograph of the HIC-HPLC run. As can be seen by thethree distinct peaks, the three antibodies are well separated and theirpeaks have little to no overlap.

Repeatability and Precision of HIC-HPLC

The repeatability is evaluated by analyzing samples of the cFDS intriplicate at 75%, 100%, 125%, 150% of the target amount (600μg/injection of cFDS). The relative standard deviation (RSD) ofrepeatability is determined to be ≤0.2%.

TABLE 4 Repeatability data Inject amount Measured Concentration [mg/mL]cFDS mAb A mAb B mAb C [μg] Ave (n = 3) RSD Ave (n = 3) RSD Ave (n = 3)RSD 450 17.4 0.1% 17 0.2% 17.9 0.2% 600 17.4 0.1% 17.3 0.1% 17.8 0.2%750 17.3 0.1% 17.4 0.2% 17.7 0.1% 900 17.1 0.1% 17.5 0.1% 17.6 0.2%

The repeatability study is repeated on a different day, using adifferent lot of column and different batch of mobile phase. The RSD ofintermediate precision is determined to be <1.7%.

TABLE 5 Intermediate precision Inject Measured Concentration [mg/mL]amount mAb A mAb B mAb C cFDS Ave Ave Ave [μg] (n = 6) RSD (n = 6) RSD(n = 6) RSD 450 17.3 0.8% 17.2 0.9% 18.0 0.7% 600 17.4 0.1% 17.3 0.2%18.0 1.1% 750 17.4 0.4% 17.4 0.2% 18.0 1.7% 900 17.1 0.1% 17.4 0.6% 17.81.6%

Linearity of HIC-HPLC

Linearity is determined by analyzing samples of cFDS samples intriplicate at 9 levels (75 to 1200 μg of cFDS; 25 to 400 μg ofindividual mAb). A plot (FIG. 10) of the peak area of individual mAbversus the injected amount results in a linear curve: At 75 to 300 μg,R²≥0.999; at 25 to 400 μg, R²≥0.98.

Range of HIC-HPLC

The range is the interval between the lowest and highest concentrationfor which the method can demonstrate acceptable precision (≤1.7%),accuracy (93-106% Recovery) and linearity (R²≥0.999). The range isdetermined to be 300 to 900 μg for cFDS sample (100 to 300 μg for eachindividual mAb). FIG. 11 is a plot of the % recovery of the mAbs vs.amount of individual mAb.

Accuracy of HIC-HPLC Across Different Lots of cFDS

Data obtained from intra-day and inter-day precision (cFDS lot #1), andrange study (cFDS lot #2) is used to evaluate the method accuracy. FIG.12 is a plot of the % recovery of the mAbs vs. amount of individual mAb.% Recovery are within 93-106% at 75%, 100%, 125%, 150% of the targetamount (600 μg total protein, 200 μg each mAb). The accuracy (%Recovery) is determined to be 93%-106%.

Accuracy of HIC-HPLC Across Various Ratios of Individual mAb in cFDS

CFDS is formulated by 1:1:1, 2:1:1, 1:2:1, 1:1:2 mix of individual mAb.Each cFDS is analyzed at 100% of the target amount (600 μg/injection,n=3). FIG. 13 is the % recovery of the mAbs vs. each individual mAb indifferent cFDS ratios. % Recovery are within 94-106% with repeatability≤0.3%.

TABLE 6 HIC-HPLC data summary Repeatability mAb A ≤0.1% (450-900 μgcFDS) (n = 3) mAb B ≤0.2% mAb C ≤0.2% Intermediate Precision mAb A ≤0.8%(450-900 μg cFDS) (n = 6) mAb B ≤0.9% mAb C ≤1.7% Accuracy 93-106% (%Recovery) (450-900 μg cFDS) Linearity R² ≥ 0.999 (a plot of the peakarea of each mAb versus the injected individual mAb amount in cFDS at 75to 300 μg range) Standard Curve R² ≥ 0.999 (50-300 μg of individual mAbstandard) Range 100 to 300 μg for each individual mAb 300 to 900 μg forcFDS sample

Application of HIC-HPLC in Co-Formulation Development

Use of HIC-HPLC to quantitate each of the mAbs in a co-formulation or aDP may have a variety of applications in co-formulation development. Forexample, the concentration of each mAb in a co-formulation or a DP maybe monitored in a storage stability study.

FIG. 14 shows a chart monitoring storage stability of three mAbs in a DPas a function of time (months). HIC-HPLC is used to quantitate theamount, and thus the concentration, of each of the three mAbs at sixpoints in time over one year. An example set of data points from onetime point in this study includes a measurement of the concentration ofmAb A at 16.6 mg/mL, the concentration of mAb B at 17.5 mg/mL, and mAb Cat 17.1 mg/mL. In this case, the storage stability shows that over thetime period monitored, there is no appreciable change in concentrationof each of the mAbs (≤4%).

The HIC-HPLC method has acceptable repeatability (≤0.2%), intermediateprecision (≤1.7%), accuracy (93%-106% recovery) and linearity (R²≥0.999)over the range of 100 to 300 μg (each individual mAb) and can be used todetermine the concentration of each of three mAbs in the cFDS and DP.

The HIC-HPLC method may be used as a release method for quantitatingeach of three mAbs in the co-formulated DS and DP. This method can alsobe used to support formulation development.

Example 4

Cation exchange ultra high-pressure liquid chromatography (CEX-UPLC)separation of a co-formulation comprising 3 anti-Ebola mAbs of similarmolecular weights, protein structures, and charge properties isdeveloped and evaluated.

The CEX-UPLC method uses a YMC-BioPro SP-F column with mobile phasesincluding 200 mM MES buffer and 10 to 120 mM NaCl gradient, with a pH of6.5. A six-point standard calibration curve is prepared for each ofthree mAb molecules (mAb A, mAb B, mAb C). The linearity of eachcalibration curve is determined to have an R²≥0.99. The three mAbmolecules are separated from a co-formulation (i.e., a mixture) usingCEX-UPLC, and a chromatograph of the UPLC is generated. Thechromatograph shows separation of each mAb, with some overlap betweenmAb A and mAb C.

The repeatability of the method is evaluated by analyzing triplicatesamples of the co-formulation run in three different amounts (150 μg,225 μg, and 300 μg). The accuracy (% recovery) is determined bycomparing the measured concentrations for each sample to the theoreticalvalue. A linear range of quantitation of each mAb is determined to be 50to 100 μg for each antibody, except for mAb A, with R²≥0.973 andaccuracy of 75%-91%.

FIG. 15 shows a chromatograph of a CEX-UPLC run of separation of 3 mAbsfrom a co-formulation. The three antibodies are separated, with a smalloverlap between mAb A and mAb C.

Repeatability and Accuracy of CEX-UPLC

The repeatability is evaluated by analyzing samples of the cFDS intriplicate at five different percentages (25%-100%) of the target amount(600 μg/injection of cFDS). The RSD of repeatability is determined to bebetween 3.7% and 21.4%

TABLE 7 Repeatability and accuracy data Inject Measured Concentration[mg/mL] amount mAb A mAb B mAb C cFDS RSD % RSD % RSD % [μg] (n = 3)Recovery (n = 3) Recovery (n = 3) Recovery 150 9.5% 80% 9.7% 83% 8.9%86% (50 per mAb) 225 3.7% 91% 5.0% 89% 3.7% 91% (75 per mAb) 300 21.4%*75% 5.2% 91% 4.9% 90% (100 per mAb) *These data points were out ofanalytical range of the method

Linearity of CEX-UPLC

Data from the repeatability study is used to evaluate the range andlinearity of the CEX-UPLC method. A plot (FIG. 16) of the peak area ofindividual mAb versus the injected amount generally results in a linearcurve for each mAb. However, because the RSD for mAb is over 10%, thedata point for mAb A at 100 μg is not sufficiently statisticallysignificant to conclude that a linear curve extending to 100 μg existsfor mAb A.

Range of CEX-UPLC

Some repeatability (RSD ≤10%), accuracy (>80%) and linearity (R²≥0.97)is shown for the cFDS sample from 150 to 300 μg in total antibodyquantity. However, a range for CEX-UPLC is not as clearly shown as forHIC-HPLC.

TABLE 8 HIC-HPLC data summary Repeatability mAb A ≤0.1% (n = 3) mAb B≤0.2% mAb C ≤0.2% Accuracy mAb A 80-91% (% Recovery) mAb B 83-91% mAb C86-91% Linearity R² ≥ 0.97 (a plot of the peak area of each mAb versusthe injected individual mAb amount in cFDS at 50 to 100 μg range)Standard Curve R² ≥ 0.99 (25-250 μg of individual mAb standard) RangemAb A  50 to 75 μg mAb B 50 to 100 μg mAb C 50 to 100 μg

The CEX-UPLC method separates three mAbs based on their chargeproperties, with some overlap. The overlap means that manual integrationof the method is needed. The precision, accuracy, and range of CEX-UPLCis not as optimal as HIC-HPLC.

Example 5

SE-UPLC of the co-formulation comprising the three anti-Ebola mAbs ofsimilar molecular weight is evaluated. SE-UPLC separates mAbs by size. AWaters Acquity H UPLC system is used. Two Waters BEH200 SEC columns arelinked in series. The mobile phase includes 10 mM Phosphate buffer at pH6.0, and 1 M Perchlorate. FIG. 17 depicts a chromatogram of the SE-UPLC.As shown, there is significant overlap between elution times of thethree mAbs. Thus, SE-UPLC is a sub-optimal method for separating thethree anti-MERS mABs of similar molecular weight.

Example 6

iCIEF of the co-formulation comprising the three anti-Ebola mAbs ofsimilar molecular weights, protein structures, and charge properties isevaluated. iCIEF separates proteins by their isoelectric point (pI). AProteinSimple iCE3 charge variant analyzer is used. 4% pH 3-10Pharmalyte® is used as an ampholyte, and 2M urea is used as a buffer.FIG. 18 depicts an iCIEF profile generated using these specifications.As shown, there is significant overlap between elution times of mAb Aand mAb C. Thus, iCIEF is a sub-optimal method for separating the threeanti-MERS mABs.

Example 7

RP-UPLC of the co-formulation comprising the three anti-Ebola mAbs ofsimilar molecular weights, protein structures, and charge properties isevaluated. RP-UPLC separates mAbs by hydrophobicity. A Waters AcquityUPLC system is used. A ZORBAX 300SB-C8 column is used, and the column isrun at 80° C. The mobile phase includes 60-90% acetonitrile in 0.1% TFA.FIG. 19 depicts a chromatogram of the RP-UPLC. As shown, there issignificant overlap between elution times of mAb A and mAb B. Thus,RP-UPLC is a sub-optimal method for separating the three anti-MERS mAbsof similar molecular weight.

EMBODIMENTS

The foregoing description discloses only exemplary embodiments of theinvention.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of theappended claims. Thus, while only certain features of the invention havebeen illustrated and described, many modifications and changes willoccur to those skilled in the art. It is therefore to be understood thatthe appended claims are intended to cover all such modifications andchanges as fall within the true spirit of the invention.

1-22. (canceled)
 23. A method of quantitating a first antibody and asecond antibody in a mixture, the method comprising: separating thefirst antibody from the second antibody by using hydrophobic interactionchromatography high performance liquid chromatography (HIC-HPLC);quantitating an amount of the first antibody; quantitating an amount ofthe second antibody; wherein each of the first antibody and the secondantibody: elutes at a distinct run time from other antibodies in themixture, when individually run on HIC-HPLC; and is a bispecificantibody.
 24. The method of claim 23, wherein the first antibody bindsto two or more proteins selected from the group consisting of: EGFR,CD28, PSMA, PD-1, CD3, CD20, and LAG3.
 25. The method of claim 24,wherein the second antibody binds to two or more target proteinsselected from the group consisting of: EGFR, CD28, PSMA, PD-1, CD3,CD20, and LAG3.
 26. The method of claim 23, wherein the first antibody,the second antibody, or both, includes a first antigen-binding arm thatspecifically binds CD20, and a second antigen-binding arm thatspecifically binds CD3.
 27. The method of claim 23, wherein during aHIC-HPLC run, the first antibody elutes at a first run time, the secondantibody elutes at a second run time, and the first and second run timesdo not overlap.
 28. The method of claim 23, wherein the first antibodyand the second antibody are human monoclonal antibodies.
 29. The methodof claim 23, wherein the first antibody and the second antibody haveprotein sequences that are at least 90% homologous; or the firstantibody and the second antibody have protein structures that are atleast 90% homologous, as determined by their protein sequences.
 30. Themethod of claim 23, wherein the mixture is a co-formulated drug product.31. The method of claim 23, wherein the co-formulated drug productfurther comprises a third antibody that binds to one or more moleculesselected from the group consisting of: GITR, LAG3, PD-1, PD-L1, EGFR,CD28, PSMA, CD3, and CD20.
 32. A method of quantitating antibodies froma co-formulated drug product comprising a plurality of antibodies, themethod comprising: generating a standard curve for each antibody of theplurality of antibodies; separating each antibody of the plurality ofantibodies by using hydrophobic interaction chromatography highperformance liquid chromatography (HIC-HPLC), wherein each antibody ofthe plurality of antibodies binds to one or more proteins selected fromthe group consisting of: GITR, LAG3, PD-1, PD-L1, EGFR, CD28, PSMA, CD3,and CD20; and after separating each antibody, quantitating an amount ofeach antibody using the standard curve.
 33. The method of claim 32,wherein one or more antibodies of the plurality of antibodies is abispecific antibody.
 34. The method of claim 32, wherein each antibodyelutes at a distinct run time from other antibodies of the plurality ofantibodies, when individually run on HIC-HPLC.
 35. The method of claim32, wherein the plurality of antibodies comprises three human monoclonalantibodies.
 36. The method of claim 32, wherein each antibody of theplurality of antibodies, has a weight within 15 kDa of every otherantibody of the plurality of antibodies.
 37. A method of quantitatingantibodies in a mixture comprising a plurality of antibodies, the methodcomprising: generating a standard curve for each antibody of theplurality of antibodies; separating each antibody of the plurality ofantibodies by using hydrophobic interaction chromatography highperformance liquid chromatography (HIC-HPLC); generating a chromatographfrom the HIC-HPLC; and quantitating an amount of each antibody of theplurality of antibodies; wherein each antibody elutes at a distinct runtime from other antibodies of the of antibodies, when individually runon HIC-HPLC; and wherein at least one antibody of the plurality ofantibodies is a bispecific antibody.
 38. The method of claim 37, whereinthe bispecific antibody comprises a first antigen-binding arm thatspecifically binds CD20, and a second antigen-binding arm thatspecifically binds CD3.
 39. The method of claim 38, wherein at least oneother antibody of the plurality of antibodies binds to GITR, LAG3, PD-1,PD-L1, EGFR, PSMA, or CD28.
 40. The method of claim 37, wherein a firstantibody, of the plurality of antibodies, binds to a first epitope of anantigen; and a second antibody, of the plurality of antibodies, binds toa second epitope of the antigen.
 41. The method of claim 37, wherein thefirst antibody and the second antibody are human monoclonal antibodies;the first antibody has a first weight, the second antibody has a secondweight, and the first weight differs from the second weight by less than15 kDa; and the first antibody and the second antibody have proteinsequences that are at least 90% homologous.
 42. The method of claim 37,further comprising, prior to generating a chromatograph, determining asurface hydrophobicity of each antibody of the plurality of antibodies,of the plurality of antibodies, wherein the surface hydrophobicity isdetermined by one or more methods selected from the group consisting of:calculated surface hydrophobicity based on protein structure orstructural model, rapid screening for solubility in ammonium sulfate orPEG8000, or rapid screening for molecule interaction by affinitycapture-self-interaction nanoparticle spectroscopy (AC-SINS).
 43. Themethod of claim 42, wherein the surface hydrophobicity is a calculatedsurface hydrophobicity based on protein structure or structural model,and a surface hydrophobicity of each antibody of the plurality ofantibodies is different from a surface hydrophobicity of anotherantibody in the mixture by more than about 1.0 unit on the Kyte &Doolittle hydropathy scale.
 44. The method of claim 37, wherein, foreach antibody of the plurality of antibodies, the chromatograph shows apeak that does not overlap with other peaks in the chromatograph.